Abstract Protein ubiquitination regulates cell biology through diverse avenues, from quality control-linked protein degradation to signaling functions such as modulating protein-protein interactions and enzyme activation. Mass spectrometry-based proteomics has allowed proteome-scale quantification of hundreds of thousands of ubiquitination sites (ubi-sites), however the functional importance and regulatory roles of most ubi-sites remain undefined. Here, we assembled a human reference ubiquitinome of 108,341 ubi-sites by harmonizing public proteomics data. We identified a core subset of ubi-sites under evolutionary constraint through alignment of ubiquitin proteomics data from six non-human species, and determined ultra-conserved ubi-sites recurring at regulatory hotspots within protein domains. Perturbation proteomics revealed that these highly conserved ubi-sites are more likely to regulate signaling functions rather than proteasomal degradation. To further prioritize functional ubi-sites with roles in cellular signaling, we constructed a functional score for more than 100,000 ubi-sites by integrating evolutionary, proteomic, and structural features using machine learning. Our score identifies ubi-sites regulating diverse protein functions and rationalizes mechanisms of genetic disease. Finally, we employed chemical genomics to validate the functional relevance of high-scoring ubi-sites and leveraged genetic code expansion to demonstrate that ubiquitination of K320 in the RNA-regulator ELAVL1 disrupts RNA binding. Our work reveals systems-level principles of the ubiquitinome and provides a powerful resource for studying protein ubiquitination.